Members of the genus Rickettsia are obligate intracytoplasmic pathogens that cause diseases such as epidemic and endemic typhus in humans. It is estimated that R. prowazekii, the louse-vectored agent of epidemic typhus, has caused more than 3 million deaths in the last century! R. prowazekii is a Select Agent and has been designated by the NIAID as a Category B Priority Pathogen. In vivo, rickettsiae are confined to a limited number of environmental niches that include transitioning between growth in gut epithelial cell of the louse vector to the blood stream of the human host and to the human endothelial cell. Considering the paucity of organisms that have successfully adapted to growth in eukaryotic cytosol as a growth niche, cytosol could be thought of as a hostile environment. In addition, due to the nature of this obligate intracellular life style, any time spent outside of a host cell under non-growing conditions is a potential stress condition indicating that the ability to communicate with the environment is likely critical to R. prowazekii pathogenesis. This R21 proposal will lay the foundation from which these extremely interesting questions can be further explored. Based on the prominent role of two-component response regulators in sensing environmental changes, I have selected the R. prowazekii ORFs RP426/RP427 (annotated as EnvZ/OmpR) as a potential sensor kinase/response regulator pair and will elucidate the regulon of genes under their control. The goal of this R21 proposal is to use this two-component network as a relevant biological model to adapt the Chromatin Immunoprecipitation (ChIP) technique for use on an obligate intracellular, cytosol-limited pathogen. The Specific Aim describes two approaches. I will use purified, recombinant OmpR, phosphorylated OmpR (OmpR approximately P) and R. prowazekii chromosomal DNA fragments to perform an in vitro ChIP assay. OmpR/OmpR approximately P-bound regulatory sequences will be isolated, cloned, and identified by DNA sequencing. This in vitro approach will identify regulatory sequences independent of environmental conditions. The second approach is an in vivo ChIP assay that will isolate regulatory DNA sequences from R. prowazekii grown under controlled environmental conditions. The conditions will be selected based on the data generated by the in vitro ChIP and also based on the limited number of environmental niches to which rickettsiae are exposed. The combination of these two techniques will elucidate R. prowazekii genes controlled by OmpR and could provide insight into the environmental stimuli to which this system responds. Consistent with the exploratory/development nature of an R21 application, this study represents the first effort to develop chromatin immunoprecipitation to elucidate a gene regulon in an obligate, intracellular pathogen using R. prowazekii as the model system. Understanding the role of gene regulation in pathogenesis may be critical to developing countermeasures and novel therapies against obligate intracellular Select Agents. [unreadable] [unreadable]